Application

Determination of Protein Turnover Rate

Protein is an important component of organisms and an essential material basis for the realization of life functions. Protein in animals is always in a dynamic equilibrium system. Protein turnover metabolism is an important dynamic process for animals to express vitality.

Protein synthesis and degradation in animals are interrelated dynamic changes, that is, while the animal is synthesizing new proteins in the body, the original tissue proteins can be degraded into amino acids that can be used for tissue protein synthesis. Waterlow used the term "protein turnover" to describe the dynamic changes in a protein in the body, which refers to the metabolic process where a protein is renewed or replaced in a particular metabolic pool. This process may result from protein synthesis or protein degradation, or may be the conversion of the same protein in different spatial distributions.

Determination of Protein Turnover Rate at Creative Proteomics

At Creative Proteomics, we have developed a professional platform to determine the protein turnover rate by using mass spectrometry as the dominant technical solution for the study of protein turnover rate for its wide availability and high resolution. Based on stable isotope labeling and mass spectrometry, a range of methodologies has been established to measure protein turnover for the proteins you are interested in, such as stable isotope labeled (SIL) tracers. First, the labeled tracer is incorporated into proteins; then proteins of interest are isolated, hydrolyzed into their amino acid constituents, and derivatized; at last, enrichment is measured via gas chromatography mass spectrometry.

Currently, there are several technical solutions to determine the turnover rates of specific proteins, including pulse-chase and bleach-chase methods. The latter is applicable to fluorescently tagged proteins, and the former is more popular because of its ease of operation, especially when stable isotopes are applied. Pulse-chase method is a technical method for determining the conversion rate of a particular protein. Labeling of cellular proteins is metabolically achieved by placing cells in a medium containing all components necessary for growth of cells in culture, except for stable isotope labeled specific amino acids for quite a short while (Pulse), and then the cell would be cultured in conventional medium with native components. The SIL amino acids would be transported across the plasma membrane and incorporated into newly synthesized proteins, which display identical physicochemical properties except molecular weight. These mass differences can be detected by high resolution mass spectrometry (Chase). By monitoring the decreasing abundance of SIL proteins, the pulse-chase technique is highly adaptable for:

Exploring the life cycle of endogenous proteins

Synthesis, folding

Subunit assembly

Intracellular transport

Post-translational processing

Degradation

With the development of mass spectrometry for proteomics research, stable isotopes such as 2H, 13C, or 15N in SIL amino acids are utilized in recent studies on protein homeostasis. The stable isotope labeling is flexible and almost applicable to all living organisms. Protein transformation defines the dynamic changes in protein expression and forms a link between transcriptome, proteome, and metabolome. Our experienced technicians and technicians are glad to discuss, select and modify agreements to provide the best procedures for your case at an affordable rate. If you have any questions or specific needs, please don’t hesitate to contact us.